Our health depends on the accurate transmission of genetic information. Multiple mutations, due to errors in DNA replication, DNA repair, and chromosome segregation, cause cancer. The need for multiple mutations selects for genetic instability, mutations that themselves increase mutation rates and thus contribute to the resistance of cancer to therapy. Studying the events that lead to genetic instability and the genes that mutate to cause instability is difficult in patients or animal models. We have made a diploid yeast model that allows us to select for mutations that improve cell proliferation by inactivating specific genes, thus leading to the evolution of genetic instability. The human homologs of genes that often mutate to cause instability in yeast will be candidate targets for mutations that cause genetic instability in human cancer and we will collaborate with a human cancer geneticist to follow these leads. The proposed work has three parts: 1) To examine the evolution of genetic instability in diploid yeast cells. Cells will be mutagenized and pools of mutant clones will be selected for stepwise inactivation of growth suppressor genes and activation of growth promoting genes. Experiments will find the mutations that cause genetic instability in 100 independently evolved examples of genetic instability, including selections for the activation of growth-promoting genes as well as the inactivation of growth suppressing genes. Preliminary results reveal mutations in genes that have not previously been implicated in genetic instability in yeast but have been implicated in human cancer. 2) For a selected subset of genes, chosen for their relevance to human cancer, more detailed experiments will examine mechanism of instability by characterizing the mutations that unstable strains produce and the mechanism of instability. Two initial examples will focus on the role of Holliday junction resolvases in stimulating mitotic recombination and determining whether different types of mutation that accelerate progress through G1 are equally like to cause genetic instability 3) Tumors are metabolically different from normal tissue and their cells are often starved. Preliminary experiments show that sudden glucose starvation leads to a rapid arrest of the yeast cell cycle and experiments will investigate how starvation arrests the yeast cell cycle and ask if this arrest or mutations that perturb the arrest lead to genetic instability.